The present invention relates to the use of modulators of stem cell proliferation for regulating stem cell cycle in the treatment of humans or animals with autoimmune diseases, aging, cancer, myelodysplasia, preleukemia, leukemia, psoriasis, acquired immune deficiency syndrome (AIDS), myelodysplastic syndromes, aplastic anemia or other diseases involving hyper- or hypo-proliferative conditions, as well as the use of such compounds for analgesia. The present invention also relates to a method of treatment for humans or animals anticipating or having undergone exposure to chemotherapeutic agents, other agents which damage cycling stem cells, or radiation exposure and for protection against such agents during ex vivo treatments. Finally, the present invention relates to the improvement of stem cell maintenance or expansion cultures for auto- and allo-transplantation procedures or for gene transfer, as well as for in vivo treatments to improve such procedures.
Most end-stage cells in renewing systems are short-lived and must be replaced continuously throughout life. For example, blood cells originate from a self-renewing population of multipotent hematopoietic stem cells (HSC). Hematopojetic stem cells are a subpopulation of hematopoietic cells. Hematopoietic cells can be obtained, for example, from bone marrow, umbilical cord blood or peripheral blood (either unmobilized or mobilized with an agent such as G-CSF); hematopoietic cells include the stem cell population, progenitor cells, differentiated cells, accessory cells, stromal cells and other cells that contribute to the environment necessary for production of mature blood cells. Hematopoietic progenitor cells are a subset of stem cells which are more restricted in their developmental potency. Progenitor cells are able to differentiate into only one or two lineages (e.g., BFU-E and CFU-E which give rise only to erythrocytes or CFU-GM which give rise to granulocytes and macrophages) while stem cells (such as CFU-MIX or CFU-GEMM) can generate multiple lineages and/or other stem cells. Because the hematopoietic stem cells are necessary for the development of all of the mature cells of the hematopoietic and immune systems, their survival is essential in order to reestablish a fully functional host defense system in subjects treated with chemotherapy or other agents.
Hematopoietic cell production is regulated by a series of factors that stimulate growth and differentiation of hematopoietic cells, some of which, for example erythropoietin, GM-CSF and G-CSF, are currently used in clinical practice. One part of the control network which has not been extensively characterized, however, is the physiological mechanism that controls the cycling status of stem cells (Eaves et al. Blood 78:110-117, 1991; Lord, in Stem Cells (C. S. Potten, Ed.) pp 401-22, 1997 (Academic Press, NY)).
Early studies by Lord and coworkers showed the existence of soluble protein factors in normal and regenerating bone marrow extracts which could either inhibit or stimulate stem cell proliferation (reviewed in: Lord and Wright, Blood Cells 6:581-593, 1980; Wright and Lorimore, Cell Tissue Kinet. 20:191-203, 1987; Marshall and Lord, Int Rev. Cyt. 167:185-261, 1996). These activities were designated stem cell inhibitor (S CT) and stem cell stimulator (SCS), respectively.
To date, no candidate SCS molecules have been purified from bone marrow extracts prepared as described by Lord et al. (reviews referenced above). Purification of either SCS or SCI from primary sources was not accomplished due to the difficulties inherent in an in vivo assay requiring large numbers of irradiated mice. In an attempt to overcome these problems Pragnell and co-workers developed an in vitro assay for primitive hematopoietic cells (CFU-A) and screened cell lines as a source of the inhibitory activity (see Graham et al. Nature 344:442-444, 1990). As earlier studies had identified macrophages as possible sources for SCI (Lord et al. Blood Cells 6:581-593, 1980), a mouse macrophage cell line, J774.2, was selected (Graham et al. Nature 344:442-444, 1990). The conditioned medium from this cell line was used by Graham et al. for purification; an inhibitory peptide was isolated which proved to be identical to the previously described cytokine macrophage inflammatory protein 1-alpha (MEP1xcex1). Receptors for MIP-1xcex1 have been cloned; like other chemokine receptors, these MIP-1xcex1 receptors are seven-transmembrane domain (or xe2x80x9cG-linkedxe2x80x9d) receptors which are coupled to guanine nucleotide (GTP) binding proteins of the Ginhibitory subclass (xe2x80x9cGixe2x80x9d) (reviewed in Murphy, Cytokine and Growth Factor Rev. 7:47-64, 1996). The xe2x80x9cinhibitoryxe2x80x9d designation for the Gi subclass refers to its inhibitory activity on adenylate cyclase.
MIP-1xcex1 was isolated from a cell line, not from primary material. While Graham et al. observed that antibody to MIP-1xcex1 abrogated the activity of a crude bone marrow extract, other workers have shown that other inhibitory activities are important. For example, Graham et al. (J. Exp. Med. 178:925-32, 1993) have suggested that TGFxcex2, not MIP-1xcex1, is a primary inhibitor of hematopoietic stem cells. Further, Eaves et al. (PNAS 90:12015-19, 1993) have suggested that both MIP-1xcex1 and TGFxcex2 are present at sub optimal levels in normal bone marrow and that inhibition requires a synergy between the two factors.
Recently, mice have been generated in which the MIP-1xcex1 gene has been deleted by homologous recombination (Cook et al., Science 269:1583-5, 1995). Such mice have no obvious derangement of their hematopoietic system, calling into question the role of MIP-1xcex1 as a physiological regulator of stem cell cycling under normal homeostatic conditions. Similarly, although transforming growth factor beta (TGFxcex2) also has stem cell inhibitory activities, the long period of time it takes for stem cells to respond to this cytokine suggests that it is not the endogenous factor present in bone marrow extracts; further, neutralizing antibodies to TGFxcex2 do not abolish SCI activity in bone marrow supernatants (Hamnpson et al., Exp. Hemat. 19:245-249, 1991).
Other workers have described additional stem cell inhibitory factors. Frindel and coworkers have isolated a tetrapeptide from fetal calf marrow and from liver extracts which has stem cell inhibitory activities (Lenfant et al., PNAS 86:779-782, 1989). Paukovits et al. (Cancer Res. 50:328-332, 1990) have characterized a pentapeptide which, in its monomeric form, is an inhibitor and, in its dimeric form, is a stimulator of stem cell cycling. Other factors have also been claimed to be inhibitory in various in vitro systems (see Wright and Pragnell in Bailliere""s Clinical Haematology v. 5, pp. 723-39, 1992 (Bailliere Tinadall, Paris); Marshall and Lord, Int Rev. Cyt. 167:185-261, 1996).
Tsyrlova et al., SU 1561261 A1, disclosed a purification process for a stem cell proliferation inhibitor.
Commonly owned applications WO 94/22915 and WO96/10634 disclose an inhibitor of stem cell proliferation, and are hereby incorporated by reference in their entirety.
To date, none of these factors have been approved for clinical use. However, the need exists for effective stem cell inhibitors. The major toxicity associated with chemotherapy or radiation treatment is the destruction of normal proliferating cells which can result in bone marrow suppression or gastrointestinal toxicity. An effective stem cell inhibitor will protect these cells and allow for the optimztion of these therapeutic regimens. Just as there is a proven need for a variety of stimulatory cytokines (i.e., cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, IL-14, IL-15, G-CSF, GM-CSF, erythropoietin, thrombopoietin, stem cell factor, flk2/flt3 ligand, etc., which stimulate the cycling of hematopoietic cells) depending upon the clinical situation, so too it is likely that a variety of inhibitory factors will be needed to address divergent clinical needs.
Further, there is a need to rapidly reverse the activity of such an inhibitor. The original studies of Lord et al. (reviews referenced above) demonstrated that the inhibitory activity could be reversed by addition of the stimulatory activity. While a variety of stem cell stimulatory cytokines has been identified (see above), none has been demonstrated to represent the activity described by Lord and coworkers as being present in bone marrow extracts and of being able to reverse the activity of the inhibitor.
Hematopoietic progenitors and stem cells primarily reside in the bone marrow in normal adults. Under certain conditions, for example chemotherapy or treatment with cytokines such as G-CSF, large numbers of progenitors and stem cells egress into the peripheral blood, a process referred to as xe2x80x9cmobilizationxe2x80x9d (reviewed in Simmons et al., Stem Cells 12 (suppl 1): 187-202, 1994; Scheding et al. Stem Cells 12 (suppl 1):203-11, 1994; Mangan, Sem. Oncology 22:202-9, 1995; Moolten, Sem. Oncology 22:271-90, 1995). Recent published data suggest that the vast majority of mobilized progenitors are not actively in cell cycle (Roberts and Metcalf, Blood 86:1600-,1995; Donahue et al., Blood 87:1644-, 1996; Siegert and Serke, Bone Marrow Trans. 17:467-1996; Uchida et al., Blood 89:465-72, 1997).
Hemoglobin is a highly conserved tetrameric protein with molecular weight of approximately 64,000 Daltons. It consists of two alpha and two beta chains. Each chain binds a single molecule of heme (ferroprotoporphyrin IX), an iron-containing prosthetic group. Vertebrate alpha and beta chains were probably derived from a single ancestral gene which duplicated and then diverged; the two chains retain a large degree of sequence identity both between themselves and between various vertebrates (see FIG. 16A). In humans, the alpha chain cluster on chromosome 16 contains two alpha genes (alpha1 and alpha2) which code for identical polypeptides, as well as genes coding for other alpha-like chains: zeta, theta and several non-transcribed pseudogenes (see FIG. 16B for cDNA and amino acid sequences of human alpha chain). The beta chain cluster on chromosome 11 consists of one beta chain gene and several beta-like genes: delta, epsilon, G gamma and A gamma, as well as at least two unexpressed pseudogenes (see FIG. 16C for cDNA and amino acid sequences of human beta chain).
The expression of these genes varies during development. In human hematopoiesis, which has been extensively characterized, embryonic erythroblasts successively synthesize tetramers of two zeta chains and two epsilon chains (Gower I), two alpha chains and two epsilon chains (Gower II) or two zeta chains and two gamma chains (Hb Portland). As embryogenesis proceeds, the predominant form consists of fetal hemoglobin (Hb F) which is composed of two alpha chains and two gamma chains. Adult hemoglobin (two alpha and two beta chains) begins to be synthesized during the fetal period; at birth approximately 50% of hemoglobin is of the adult form and the transition is complete by about 6 months of age. The vast majority of hemoglobin (approximately 97%) in the adult is of the two alpha and two beta chain variety (Hb A) with small amounts of Hb F or of delta chain (Hb A2) being detectable.
Several methods have been used to express recombinant hemoglobin chains in E. coli and in yeast (e.g., Sessen et al., Methods Enz. 231:347-364, 1994; Looker et al., Methods Enz. 231:364-374, 1994; Ogden et al., Methods Enz. 231:374-390, 1994; Martin de Llano et al., Methods Enz. 231:364-374, 1994). It has thus far not been possible to express isolated human alpha chain in high yields by recombinant methods (e.g., Hoffman et al., PNAS 87:8521-25, 1990; Heman et al., Biochem. 31:8619-28, 1992). Apparently, the isolated alpha chain does not assume a stable conformation and is rapidly degraded in E. coli. Co-expression of beta chain with alpha chain results in increased expression of both (Hoffman et al. and Hernan et al., op. cit.). While the alpha chain has been expressed as a fusion protein with a portion of the beta chain and a factor Xa recognition site (Nagai and Thorgersen, Methods Enz. 231:347-364, 1994) it is expressed as an insoluble inclusion body under these conditions.
Both the beta chain and the alpha chain contain binding sites for haptoglobin. Haptoglobin is a serum protein with extremely high affinity for hemoglobin (e.g., Putnam in The Plasma Proteinsxe2x80x94Structure, Function and Genetic Control (F. W. Putnam, Ed.) Vol. 2, pp 1-49 (Academic Press, NY); Hwang and Greer, JBC 255:3038-3041, 1980). Haptoglobin transport to the liver is the major catabolic pathway for circulating hemoglobin. There is a single binding site for haptoglobin on the alpha chain (amino acids 121-127) and two on the beta chain (amino acid regions 11-25 and 131-146) (Kazim and Atassi, Biochem J. 197:507-510, 1981; McCormick and Atassi, J. Prot Chem. 9:735-742, 1990).
Biologically active peptides with opiate activity have been obtained by proteolytic degradation of hemoglobin (reviewed in Karelin et al., Peptides 16:693-697, 1995). Hemoglobin alpha chain has an acid-labile cleavage site between amino acids 94-95 (Shaeffer, J. Biol. Chem. 269:29530-29536, 1994).
Kregler et al. (Exp. Hemat. 9:11-21, 1981) have disclosed that hemoglobin has an enhancing activity on mouse bone marrow CFU-C progenitor colonies. Such assays demonstrate effects on CFU-GM and CFU-M progenitor populations as opposed to stem cells such as CFU-MIX. The authors observed activity in both isolated alpha and beta chains of hemoglobin. This activity was abolished by treatment with N-ethylmaleimide, which suggested to Kregler et al. that sulfhydryl groups were necessary. This observation, coupled with the fact that the stimulatory activity was resistant to trypsin digestion, suggested to Kregler et al. that the C-terminal hydrophobic domain or xe2x80x9ccorexe2x80x9d region was responsible for the activity. Moqattash et al. (Acta. Haematol. 92:182-186, 1994) have disclosed that recombinant hemoglobin has a stimulatory effect on CFU-E, BFU-E and CFU-GM progenitor cell number which is similar to that observed with hemin. Mueller et al. (Blood 86:1974, 1995) have disclosed that purified adult hemoglobin stimulates erythroid progenitors in a manner similar to that of hemin.
Petrov et al. (Bioscience Reports 15:1-14, 1995) disclosed the use of a xe2x80x9cnonidentified myelopeptide mixturexe2x80x9d in the treatment of congenital anemia in the Wv/Wv mouse. The mixture increased the number of spleen colonies, especially those of the erythroid type.
Heme and hemin have been extensively examined with regard to their influences on hematopoiesis (see S. Sassa, Seminars Hemat. 25:312-20, 1988 and N. Abraham et al., Int. J. Cell Cloning 9:185-210, 1991 for reviews). Heme is required for the maturation of erythroblasts; in vitro, hemnin (chloroferroprotoporphyrin IXxe2x80x94i.e., heme with an additional chloride ion) increases the proliferation of CFU-GEMM, BFU-E and CFU-E. Similarly, hemin increases cellularity in long-term bone marrow cultures.
xe2x80x9cOpiatesxe2x80x9d are substances with analgesic properties similar to morphine, the major active substance in opium. Opiates can be small organic molecules, such as morphine and other alkaloids or synthetic compounds, or endogenous peptides such as enkephalins, endorphins, dynorphins and their synthetic derivatives. Endogenous opiate peptides are produced in vivo from larger precursorsxe2x80x94pre-proenkephalin A for Met- and Leu-enkephalins, pre-proopiomelanocortin for xcex1, xcex2, and xcex3 endorphins, and pre-prodynorphin for dynorphins A and B, xcex1-neoendorphin and xcex2-neoendorphin. In addition, peptides with opiate activity can be obtained from non-classical sources such as proteolysis or hydrolysis of proteins such as xcex1 or xcex2 casein, wheat gluten, lactalbumin, cytochromes or hemoglobin, or from other species such as frog skin (dermorphins) or bovine adrenal medulla. Such peptides have been termed xe2x80x9cexorphinsxe2x80x9d in contrast to the classical endorphins; they are also referred to as atypical opiate peptides (Zioudrou et al., JBC 254:2446-9, 1979; Quirion and Weiss, Peptides 4:445, 1983; Loukas et al., Biochem. 22:4567, 1983; Brantl, Eur. J. Pharm. 106:213-14, 1984; Brantl et al., Eur. J. Pharm. 111:293-4, 1985; Brand et al., Eur. J. Pharm. 125:309-10, 1986; Brantl and Neubert, TIPS 7:6-7,1986; Glamsta et al., BBRC 184:1060-6, 1992; Teschemacher, Handbook Exp. Pharm. 104:499-28, 1993; Petrov et al., Bioscience Reports 15:1-14, 1995; Karelin et al., Peptides 16:693-7, 1995). Other endogenous peptides, such as the Tyr-MIF-1 family, have also been shown to have opiate activity (Reed et al., Neurosci. and Biobehav. Rev. 18:519-25, 1994).
Opiates exert their actions by binding to three main pharmacological classes of endogenous opiate receptorsxe2x80x94mu, delta, and kappa. Receptors representing each pharmacological class have been cloned and shown to be G-liked receptors coupled to Gi (reviewed in: Reisine and Bell, TINS 16: 506-510, 1993; Uh1 et al., TINS 17:89-93, 1994: Knapp et al., FASEB J. 9:516-525, 1995; Satoh and Minami, Pharm. Ther. 68:343-64, 1995; Kieffer, Cell. Mol. Neurobiol. 15:615-635, 1995: Reisine, Neuropharm. 34:463-472, 1995; Zaki et al., Ann. Rev. Pharm. Toxicol., 36:379-401, 1996).
Specific agonists and antagonists are available for each receptor typexe2x80x94e.g., for mu receptors (which are selectively activated by DAMGO and DALDA and selectively antagonized by CTOP and naloxonazine), for kappa receptors (which are selectively activated by GR 89696 fumarate or U-69593 and selectively antagonized by norxe2x80x94binaltorphimine hydrochloride) and for delta receptors (which are selectively activated by DADLE and DPDPE and selectively antagonized by natrindole). In addition, there are broad-spectrum antagonists (such as naloxone) and agonists (such as etorphine) which act on all three receptor subtypes.
Both classical and atypical opiate peptides can be chemically altered or derivatized to change their specific opiate receptor binding properties (reviewed in Hruby and Gehrig, Med. Res. Rev. 9:343-401, 1989; Schiller, Prog. Med. Chem. 28: 301-40, 1991; Teschemacher, Handbook Exp. Pharm. 104:499-28, 1993; Handbook of Experimental Pharmacology, A. Hertz (Ed.) volumes 104/I and 104/II, 1993, Springer Verlag, Berlin; Karelin et al., Peptides 16:693-7, 1995). Examples include derivatives of dermorphin (e.g., DALDA) and enkephalins (e.g., DADLE, DAMGO or DAMME). Peptides which do not normally bind to opiate receptors, such as somatostatin, can also be derivatized to exhibit specific opiate receptor binding (e.g., CTOP (Hawkins et al., J. Pharm. Exp. Ther. 248:73, 1989)). Analogs can also be derived from alkaloids such as morphine with altered receptor binding properties (e.g., heroin, codeine, hydromorphone, oxymorphone, levorphanol, levallorphan, codeine, hydrocodone, oxycodone, nalorphine, naloxone, naltrexone, buprenorphine, butanorphanol and nalbuphine); in addition, small molecules structurally unrelated to morphine can also act on opiate receptors (e.g., meperidine and its congeners alphaprodine, diphenoxylate and fentanyl) (see Handbook of Experimental Pharmacology, op. cit.; Goodman and Gilman""s The Pharmacological Basis of Therapeutics, 7th Ed., A. G. Gilman, L. S. Goodman, T. W. Rall and F. Murad (Eds.) 1985 Macmillan Publishing Co. NY).
The endogenous opiate peptides (enkephalins, endorphins and dynorphins) have a conserved N-terminal tetrapeptide Tyr-Gly-Gly-Phe, followed by Leu or Met and any remaining C-terminal sequence. Removal of the hydroxyl group on the N-terminal Tyr (resulting in an N-terminal Phe) results in a dramatic loss of activity for Met-enkephalin. These structural data led to the xe2x80x9cmessage-addressxe2x80x9d hypothesis whereby the N-terminal xe2x80x9cmessagexe2x80x9d confers biological activity while the C-terminal xe2x80x9caddressxe2x80x9d confers specificity and enhanced potency (Chavkin and Goldstein, PNAS 78:6543-7, 1981). Exorphins generally have a Tyr-Pro replacing the N-terminal Tyr-Gly of classical opiate peptides; the proline residue is thought to confer higher stability against aminopeptidase degradation (Shipp et al., PNAS 86: 287-, 1989; Glamsta et al., BBRC 184:1060-6, 1992).
Recently an orphan receptor (xe2x80x9cORL1xe2x80x9d) was cloned by virtue of sequence relatedness to the mu, delta and kappa opiate receptors (Mollereau et al., FEBS 341:33-38, 1994; Fukuda et al., FEBS 343:42-46, 1994; Bunzow et al., FEBS 347:284-8, 1994; Chen et al., FEBS 347:279-83, 1994; Wang et al., FEBS 348:75-79, 1994; Keith et al., Reg. Peptides 54 143-4, 1994; Wick et al., Mol. Brain Res. 27: 37-44, 1994, Halford et al., J. Neuroimmun. 59:91-101, 1995). The ligand for this receptor, variously called nociceptin or orphanin FQ (referred to hereafter as xe2x80x9cnociceptinxe2x80x9d) has been cloned and shown to be a heptadecapeptide which is derived from a larger precursor (Meunier et al., Nature 377:532-535, 1995; Reinscheid et al., Science 270:792-794, 1995). It was demonstrated to have pronociceptive, hyperalgesic functions in vivo, as opposed to classical opiates which have analgesic properties. Nociceptin has a Phe-Gly-Gly-Phe . . . N-terminal motif in contrast to the Tyr-Gly-Gly-Phe . . . N-terminal motif of classical opiate peptides discussed above. In keeping with the requirement for an N-terminal Tyr for opiate activity in classical opiate peptides, nociceptin exhibits little or no affinity for the mu, kappa or delta opiate receptors. Similarly, the broad-spectrum opiate antagonist naloxone has no appreciable affinity for ORL1.
Enkephalins have been observed to have effects on murine hematopoiesis in vivo under conditions of immobilization stress (Goldberg et al., Folia Biol. (Praha) 36:319-331, 1990). Leu-enkephalin inhibited and met-enkephalin stimulated bone marrow hematopoiesis. These effects were indirect, Goldberg et al. believed, due to effects on glucocorticoid levels and T lymphocyte migration. Krizanac-Bengez et al. (Biomed. and Pharmacother. 46:367-43, 1992; Biomed. and Pharmacother. 49:27-31, 1995; Biomed. and Pharmacother. 50:85-91, 1996) looked at the effects of these compounds in vitro. Pre-treatment of murine bone marrow with Met- or Leu-enkephalin or naloxone affected the number of GM progenitor cells observed in a colony assay. This effect was highly variable and resulted in suppression, stimulation or no effect; further, there was no clear dose-response. This variability was ascribed by Krizanac-Bengez et al. to circadian rhythms and to accessory cells.
Recently, it has been demonstrated that mice in which the mu opiate receptor has been deleted by homologous recombination have elevated numbers of CFU-GM, BFU-E and CFU-GEMM per femur. Marrow and splenic progenitors were more rapidly cycling in these mu receptor knockout mice compared to normal mice. It was not determined if these effects were due to a direct or indirect effect on bone marrow stem cells resulting from the absence of the mu receptor in these animals (Broxmeyer et al., Blood 88:338a, 1997).
Productive research on stimulatory growth factors has resulted in the clinical use of a number of these factors (erythropoietin, G-CSF, GM-CSF, etc.). These factors have reduced the mortality and morbidity associated with chemotherapeutic and radiation treatments. Further clinical benefits to patients who are undergoing chemotherapy or radiation could be realized by an alternative strategy of blocking entrance of stem cells into cell cycle thereby protecting them from toxic side effects. The reversal of this protection will allow for rapid recovery of bone marrow function subsequent to chemo- or radiotherapy.
Bone marrow transplantation (BMT) is a useful treatment for a variety of hematological, autoirmune and malignant diseases. Current therapies include hematopoietic cells obtained from umbilical cord blood, fetal liver or from peripheral blood (either unmobilized or mobilized with agents such as G-CSF or cyclophosphamide) as well as from bone marrow; the stem cells may be unpurified, partially purified (e.g., affinity purification of the CD34+ population) or highly purified (e.g., through fluorescent activated cell sorting using markers such as CD34, CD38 or rhodamine). Ex vivo manipulation of hematopoietic cells is currently being used to expand primitive stem cells to a population suitable for transplantation. Optimization of this procedure requires: (1) sufficient numbers of stem cells able to maintain long term reconstitution of hematopoiesis; (2) the depletion of graft versus host-inducing T-lymphocytes and (3) the absence of residual malignant cells. This procedure can be optimized by including a stem cell inhibitor(s) and/or a stem cell stimulator(s).
The effectiveness of purging of hematopoietic cells with cytotoxic drugs in order to eliminate residual malignant cells is limited due to the toxicity of these compounds for normal hematopoietic cells and especially stem cells. There is a need for effective protection of normal cells during purging; protection can be afforded by taking stem cells out of cycle with an effective inhibitor.
Peripheral blood stem cells (PBSC) offer a number of potential advantages over bone marrow for autologous transplantation. Patients without suitable marrow harvest sites due to tumor involvement or previous radiotherapy can still undergo PBSC collections. The use of blood stem cells eliminates the need for general anesthesia and a surgical procedure in patients who would not tolerate this well. The apheresis technology necessary to collect blood cells is efficient and widely available at most major medical centers. The major limitations of the method are both the low normal steady state frequency of stem cells in peripheral blood and their high cycle status after mobilization procedures with drugs or growth factors (e.g., cyclophosphamide, G-CSF, stem cell factor). An effective stem cell inhibitor will be useful to return such cells to a quiescent state, thereby preventing their loss through differentiation.
A number of diseases are characterized by a hyperproliferative state in which dysregulated stem cells give rise to an overproduction of end stage cells. Such disease states include, but are not restricted to, psoriasis, in which there is an overproduction of epidermal cells, premalignant conditions in the gastrointestinal tract characterized by the appearance of intestinal polyps, and acquired immune deficiency syndrome (AIDS) where early stem cells are not infected by HIV but cycle rapidly resulting in stem cell exhaustion. A stem cell inhibitor will be useful in the treatment of such conditions.
A number of diseases are characterized by a hypoproliferative state in which dysregulated stem cells give rise to an underproduction of end stage cells. Such disease states include myelodysplatic syndromes or aplastic anemia, in which there is an underproduction of blood cells, and conditions associated with aging where there is a deficiency in cellular regeneration and replacement A stem cell stimulator will be useful in the treatment of such conditions.
The ability to transfer genetic information into hematopoietic cells is currently being utilized in clinical settings. Hematopoietic cells are a useful target for gene therapy because of ease of access, extensive experience in manipulating and treating this tissue ex vivo and because of the ability of blood cells to permeate tissues. Furthermore, the correction of certain human genetic defects can be possible by the insertion of a functional gene into the primitive stem cells of the human hematopoietic system.
There are several limitations for the introduction of genes into human hematopoietic cells using either retrovirus vectors or physical techniques of gene transfer: (1) The low frequency of stem cells in hematopoietic tissues has necessitated the development of high efficiency gene transfer techniques; and (2) more rapidly cycling stem cells proved to be more susceptible to vector infection, but the increase of the infection frequency by stimulation of stem cell proliferation with growth factors produces negative effects on long term gene expression, because cells containing the transgenes are forced to differentiate irreversibly and lose their self-renewal. These problems can be ameliorated by the use of a stem cell inhibitor to prevent differentiation and loss of self-renewal and a stem cell stimulator to regulate the entry of stem cells into cycle and thereby facilitate retroviral-mediated gene transfer.
The present invention relates to compounds including peptides and polypeptides which are inhibitors and/or stimulators of stem cell proliferation (INPROL and opiate compounds) and their use.
The present invention includes an inhibitor of stem cell proliferation characterized by the following properties:
(a) Specific activity (IC50) less than or equal to 20 ng/ml in a murine colony-forming spleen (CFU-S) assay (see Example 4),
(b) Molecular weight greater than 10,000 and less than 100,000 daltons (by ultrafiltration),
(c) Activity sensitive to degradation by trypsin,
(d) More hydrophobic than MIP-1xcex1 or TGFxcex2 and separable from both by reverse phase chromatography (see Example 12),
(e) Biological activity retained after heating for one hour at 37xc2x0 C., 55xc2x0 C. or 75xc2x0 C. in aqueous solution and
(f) Biological activity retained after precipitation with 1% hydrochloric acid in acetone.
The present invention is further characterized and distinguished from other candidate stem cell inhibitors (e.g., MIP-1xcex1, TGFxcex2 and various oligopeptides) by its capacity to achieve inhibition in an in vitro assay after a short preincubation period (see Example 5).
The present invention also comprises pharmaceutical compositions containing INPROL for treatment of a variety of disorders.
The present invention provides a method of treating a subject anticipating exposure to an agent capable of killing or damaging stem cells by administering to that subject an effective amount of a stem cell inhibitory composition. The stem cells protected by this method can be hematopoietic stem cells ordinarily present and dividing in the bone marrow, cord blood, fetal liver or mobilized into the peripheral blood circulation. While the majority of mobilized stem cells are quiescent according to fluorescence activated cell sorter (FACS) analysis, the multipotential stem cells are demonstrated to be cycling and inhibitable by INPROL at stem cell inhibitory amounts. Alternatively, stem cells can be epithelial, located for example, in the intestines or scalp or other areas of the body or germ cells located in reproductive organs. The method of this invention can be desirably employed on humans, although animal treatment is also encompassed by this method. As used herein, the terms xe2x80x9csubjectxe2x80x9d or xe2x80x9cpatientxe2x80x9d refer to an animal, such as a mammal, including a human.
The present invention also provides a method of treating a subject with hypoproliferating stem cells by administering to that subject an effective amount of a stem cell stimulatory composition. The stem cells stimulated by this method can be hematopoietic stem cells ordinarily present in the bone marrow, cord blood, fetal liver or mobilized into the peripheral blood circulation; such stem cells may have previously been placed into quiescence by use of INPROL at stem cell inhibitory amounts. INPROL at stem cell stimulatory amounts will allow for stimulation of stem cell cycling when desiredxe2x80x94for example, after harvesting of stem cells for use during ex vivo expansion, or in vivo subsequent to stem cell transplantation and engraftment. Alternatively, stem cells can be epithelial, located for example, in the intestines, or scalp or other areas of the body or germ cells located in reproductive organs.
In another aspect, the invention provides a method for protecting and restoring the hematopoietic, immune or other stem cell systems of a patient undergoing chemotherapy, which includes administering to the patient an effective stem cell inhibitory amount of INPROL and/or to stimulate recovery after chemotherapy or radiation by administering an effective stem cell stimulatory amount of INPROL.
In still a further aspect, the present invention involves a method for adjunctively treating any cancer, including those characterized by solid tumors (e.g., breast, colon, lung, testicular, ovarian, liver, kidney, pancreas, brain, sarcoma), by administering to a patient having cancer an effective stem cell inhibitory amount of INPROL to protect stem cells of the bone marrow, gastrointestinal tract or other organs from the toxic effects of chemotherapy or radiation therapy and/or to stimulate recovery after chemotherapy or radiation therapy by administering stem cell stimulatory amounts of INPROL.
Yet another aspect of the present invention involves the treatment of leukemia (e.g., chronic myelogenous leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia, myeloma, Hodgkin""s disease), comprising treating hematopoietic cells having proliferating leukemia cells therein with an effective amount of INPROL to inhibit proliferation of normal stem cells, and treating the bone marrow with a cytotoxic agent to destroy leukemia cells. This method can be enhanced by the follow-up treatment of the bone marrow with other agents that stimulate its proliferation; e.g., colony stimulating factors and/or INPROL at stem cell stimulatory amounts. In one embodiment this method is performed in vivo. Alternatively, this method is also useful for ex vivo purging and expansion of hematopoietic cells for transplantation.
In still a further aspect, the method involves treating a subject having any disorder caused by proliferating stem cells. Such disorders, such as psoriasis, myelodysplasia, some autoimmune diseases, immuno-depression in aging, myelodysplastic syndrome, aplastic anemia or stem cell exhaustion in AIDS are treated by administering to the subject an effective amount of INPROL to inhibit or to stimulate proliferation of the stem cell in question.
The present invention provides a method for reversibly protecting stem cells from damage from a cytotoxic agent capable of killing or damaging stem cells. The method involves administering to a subject anticipating exposure to such an agent an effective stem cell inhibitory amount of INPROL.
The present invention also provides a method for reversibly stimulating the proliferation of stem cells during the recovery phase after chemotherapy or radiation. The method involves administering to a subject anticipating exposure to such an agent, an effective stem cell stimulatory amount of INPROL.
The present invention also provides:
An inhibitor of stem cell proliferation isolated from porcine or other bone marrow by the following procedure (see Example 12):
(a) Extraction of bone marrow and removal of particulate matter through filtration,
(b) Heat treatment at 56xc2x0 C. for 40 minutes followed by cooling in ice bath,
(c) Removal of precipitate by centrifugation at 10,000 g for 30 minutes at 4xc2x0 C.,
(d) Acid precipitation by addition of supernatant to 10 volumes of stirred ice-cold acetone containing 1% by volume concentrated hydrochloric acid and incubation at 4xc2x0 C. for 16 hours,
(e) Isolation of precipitate by centrifugation at 20,000 g for 30 minutes at 4xc2x0 C. and washing with cold acetone followed by drying,
(f) Isolation by reverse phase chromatography and monitoring activity by inhibition of colony formation by bone marrow cells pretreated with 5-fluorouracil and incubated in the presence of murine IL-3, as well as by absorption at 280 nm and by SDS-PAGE.
The present invention also provides:
A method for purifying an inhibitor of stem cell proliferation substantially free from other proteinaceous materials comprising the preceding steps, as also described in more detail below.
The present invention also provides:
A method of treatment for humans or animals wherein an inhibitor of stem cell proliferation functions to ameliorate immunosuppression caused by stem cell hyperproliferation.
The present invention also provides:
A method of treatment for humans or animals wherein INPROL at stem cell stimulatory amounts ameliorates bone marrow suppression caused by stem cell hypoproliferation.
The present invention also provides:
A method of treatment for humans or animals wherein said inhibitor of stem cell proliferation is administered after the stem cells are induced to proliferate by exposure to a cytotoxic drug or irradiation procedure. Stem cells are normally quiescent but are stimulated to enter cell cycle after chemotherapy. This renders them more sensitive to a second administration of chemotherapy; the current method protects them from this treatment
The present invention also provides:
A method of treatment for humans or animals wherein a stimulator of stem cell proliferation (e.g., INPROL at stem cell stimulatory amounts) is administered, before or after INPROL at stem cell inhibitory amounts, to promote bone marrow regeneration. Stem cell inhibitory amounts of INPROL slow the rate at which stem cells transit the cell cycle and protect against chemotherapy or radiation; stem cell stimulatory amounts of INPROL reverse this inhibition and promote bone marrow recovery. Conversely, stem cell stimulatory amounts of INPROL can be used to promote bone marrow recovery while stem cell inhibitory amounts are used subsequently to return stem cells to quiescence once bone marrow recovery is achieved.
The present invention also provides:
A method of treatment for humans or animals wherein said inhibitor of stem cell proliferation is administered as an adjuvant before or together with vaccination for the purpose of increasing immune response.
The present invention also provides:
A method of treating immune deficiency in a mammal comprising administering to said mammal an immunostimulatory amount of INPROL.
The present invention also provides:
A method of treating pain in a mammal comprising administering to said mammal an analgesia-inducing amount of INPROL.
The present invention also provides:
A method of treatment for humans or animals receiving cytotoxic drugs or radiation treatment which comprises administering an effective amount of the inhibitor of stem cell proliferation to protect stem cells against damage.
The present invention also provides:
A method of treatment for humans or animals receiving cytotoxic drugs or radiation treatment which comprises administering an effective stem cell stimulatory amount of INPROL to enhance recovery after treatment.
The invention also includes a pharmaceutical composition comprising hemoglobin and a pharmaceutically acceptible carrier.
The invention also includes a pharmaceutical composition comprising (a) a polypeptide selected from the group consisting of the alpha chain of hemoglobin, the beta chain of hemoglobin, the gamma chain of hemoglobin, the delta chain of hemoglobin, the epsilon chain of hemoglobin and the zeta chain of hemoglobin, the polypeptide comprising amino acids 1-97 of the human alpha hemoglobin chain (xe2x80x9cpeptide 1-97xe2x80x9d) and the polypeptide comprising amino acids 1-94 of the human alpha hemoglobin chain (xe2x80x9cpeptide 1-94xe2x80x9d) and (b) a pharmaceutically acceptible carrier. Such pharmaceutical compositions be can composed of a single polypeptide selected from said group, a mixture of polypeptides selected from said group or polypeptides from said group in the form of dimers or multimers, with or without heme.
The invention also includes peptides having the sequences:
Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val (Seq ID No:1)
(xe2x80x9cPeptide 43-55xe2x80x9d),
Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys (Seq ID No:2)
where the two Cys residues form a disulfide bond
(xe2x80x9cCyclic Peptide 43-55xe2x80x9d),
Cys-Phe-Pro-His-Phe-Asp-Leu-Ser-His-Gly-Ser-Ala-Gln-Val-Cys
where the two Cys residues are joined by a carbon bridge,
Asp-Ala-Leu-Thr-Asn-Ala-Val-Ala-His-Val-Asp-Asp-Met-Pro-Asn-Ala-Leu-Ser-Ala (Seq ID No:3)
(xe2x80x9cPeptide 64-82xe2x80x9d), and
a peptide comprising the first 97 N-terminal amino acids of human alpha hemoglobin as in FIG. 16A.
Also included in the invention are proteins and peptide sequences consisting of modified versions of the human alpha chain which retain stem cell inhibitory and/or stimulatory properties. Such modifications include, but are not limited to, removal or modification of the C-terminal hydrophobic domain (resulting in improved solubility characteristics) and/or removal or modification of the haptoglobin binding domain (resulting in improved pharmacokinetic properties). The C-terminal hydrophobic domain in human alpha hemoglobin is comprised of the region from amino acids 98 (phenylalanine) to 141 (arginine) and contains 23 hydrophobic amino acids Out of a total of 44. The entire domain or one or more of these hydrophobic amino acids (6 alanines, 4 valines, 8 leucines, 2 proline and 3 phenylalanines) can be removed by deletion (xe2x80x9cdeletedxe2x80x9d C-terminal hydrophobic domain). Alternatively, one or more of these amino acids can be substituted with a non-polar amino acid (e.g., glycine, serine, threonine, cysteine, tyrosine, asparagine or glutamine) (xe2x80x9csubstitutedxe2x80x9d C-terminal hydrophobic domain).
In another embodiment, chemical modifications such as carboxymethylation, which decrease the hydrophobic character of this region and increases solubility, is used.
In another embodiment, hydrophobic residues are substituted with the corresponding hydrophilic regions in the human beta hemoglobin sequence. For example, in the human beta hemoglobin sequence, the region between amino acids 107 (glycine) to 117 (histidine) or the region between amino acids 130 (tyrosine) to 139 (asparagine) are each relatively hydrophilic and each or both can be substituted for the equivalent hydrophobic regions in human alpha hemoglobin.
The haptoglobin binding domain is contained within the C-terminal hydrophobic region and is comprised of amino acids 121-127. This region can be removed by deletion in its entirety or one or more amino acids in this region can be deleted (xe2x80x9cdeletedxe2x80x9d C-terminal haptoglobin binding domain). This region or one or more amino acids in this region can be substituted with other amino acids such as, for example, poly-alanine or poly-glycine or other amino acids which have the effect of abolishing the binding of the polypeptide to haptoglobin but maintain the stem cell inhibitory activity (xe2x80x9csubstitutedxe2x80x9d C-terminal haptoglobin binding domain).
Other embodiments of the invention encompass corresponding modifications to the beta hemoglobin chain (either in the C-terminal hydrophobic region and/or in one or both haptoglobin binding domains (amino acids 11-25 and 136-146)), and corresponding modifications to the delta, gamma, epsilon and/or zeta hemoglobin chains.
Also included in the invention are DNA sequences encoding the above identified peptides, vectors containing said DNA sequences and host cells containing said vectors. These peptides can be synthesized using standard chemical techniques (e.g., solid phase synthesis) or by using recombinant techniques (including fusion systems such as those employing glutathione-S-transferase (D. B. Smith and K. S. Johnson, Gene 67:31-40, 1988), thioredoxin (LaVallie et al., Biotechnology 11:187-193, 1993) or ubiquitin (Butt et al., PNAS 86:2540-4, 1989; Cherney et al., Biochem. 30:10420-7, 1991; Baker et al., JBC 269:25381-6, 1994; U.S. Pat. Nos. 5,132,213; 5,196,321 and 5,391,490 and PCT WO 91/17245). Each of these articles, applications and patents is hereby incorporated by reference.
Additionally the invention includes a method of inhibiting or stimulating stem cell proliferation comprising contacting hematopoietic cells with a compound capable of binding opiate receptors, advantageously the mu subclass of opiate receptors. Additionally the invention includes a method of inhibiting or stimulating stem cell proliferation comprising contacting hematopoietic cells with a compound capable of binding nociceptin receptors (e.g., ORL1). Further, the invention includes a method of inhibiting or stimulating stem cell proliferation comprising contacting hematopoietic cells with a compound capable of binding xe2x80x9copiate-likexe2x80x9d receptors.
Peptides (called xe2x80x9chemorphinsxe2x80x9d) have been isolated from hemoglobin which exhibit opiate activities (e.g., Brantl et al., Eur. J. Pharm, 125:309-10, 1986; Davis et al. Peptides 10:747-51, 1989; Hoffman et al., PNAS 87:8521-25, 1990; Hernan et al., Biochem. 31:8619-28, 1992; Karelin et al. Bioch. Biophys. Res. Comm, 202:410-5, 1994; Zhao et al., Ann. N.Y. Acad. Science 750:452-8, 1995; Petrov et at., Bioscience Reports, 15:1-14, 1995; Karelin et al., Peptides 16:693-697, 1995). Each of these articles is hereby incorporated by reference. Other atypical opiate peptides and small molecules also exist (Zioudrou et al., JBC 254:2446-9,1979; Quirion and Weiss, Peptides 4:445, 1983; Loukas et al., Biochem. 22:4567, 1983; Brantl, Eur. J. Pharm. 106:213-14, 1984; Brantl et al., Eur. J. Pharm. 111:293-4, 1985; Brand and Neubert, TIPS 7:6-7,1986; Hruby and Gehrig, Med. Res. Rev. 9:343-401, 1989; Schiller, Prog. Med. Chem. 28: 301-40, 1991; Glamsta et al., BBRC 184:1060-6, 1992; Teschemacher, Handbook Exp. Pharm. 104:499-28, 1993; Handbook of Experimental Pharmacology, A. Hertz (Ed.) volumes 104/I and 104/II, 1993, Springer Verlag, Berlin; Reed et al., Neurosci. and Biobehav. Rev. 18:519-25, 1994; Karelin et al., Peptides 16:693-7, 1995). Each of these articles is hereby incorporated by reference. As used herein, xe2x80x9copiate-like receptorsxe2x80x9d are defined by their ability to bind opiates, INPROL, hemorphins, exorphins, nociceptin, Tyr-MIF-1 family members, alkaloids and/or other compounds which either inhibit or stimulate stem cell proliferation in a manner antagonized by the inclusion of an appropriate amount of naloxone (see Examples 29 and 38).
In addition, the invention includes a method of identifying receptor(s) and ligands comprising using INPROL (advantageously peptide forms such as Peptide 1-94, 1-97, 43-55 or 64-82) in a receptor binding assay. Further, the invention includes a method of identifying receptor(s) and ligands comprising using INPROL in an adenylate cyclase assay.
Additionally the invention includes a method of inhibiting or stimulating stem cell proliferation comprising contacting hematopoietic cells with a compound (for example, mastoparan) capable of activating GTP-binding proteins, advantageously those of the Ginhibitory subtype.
The invention also includes a method of inhibiting or stimulating stem cell proliferation comprising contacting hematopoietic cells with a peptide selected from the group of hemorphin peptides having the sequence:
Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe, (SEQ ID NO:4);
Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg, (SEQ ID NO:5);
Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln, (SEQ ID NO:6);
Leu-Val-Val-Tyr-Pro-Trp-Thr, (SEQ ID NO:7);
Leu-Val-Val-Tyr-Pro-Trp, (SEQ ID NO:8);
Leu-Val-Val-Tyr-Pro, (SEQ ID NO:9);
Val-Val-Tyr-Pro-Trp-Thr-Gln, (SEQ ID NO:10);
Tyr-Pro-Trp-Thr-Gln-Arg-Phe, (SEQ ID NO:11);
Tyr-Pro-Trp-Thr-Gln-Arg, (SEQ ID NO:12);
Tyr-Pro-Trp-Thr-Gln, (SEQ ID NO:13); and
Tyr-Pro-Trp-Thr (SEQ ID NO:27).
The above peptides have sequence similarity and/or biological activity similar to other atypical opiate peptides such as those of the Tyr-MIF-1 family (see Reed et al., Neurosci. Biobehav. Rev. 18:519-25, 1994 for review), the casein-derived casomorphins (Branl et al., Hoppe-Seyler""s Z. Physiol. Chem. 360:1211-16, 1979; Loukas et al., Biochem. 22:4567-4573, 1983; Fiat and Jolles, Mol. Cell. Biochem. 87:5-30, 1989), peptides derived from cytochrome b, termed cytochrophins (Brantl et al., Eur. J. Pharm. 111:293-4, 1985), various exorphins and opiate peptides from human and non-human species (Zioudrou et al., JBC 254:2446-9, 1979; Brantl, Eur. J. Pharm. 106:213-14, 1984; Branl et al., Eur. J. Pharm. 125:309-10, 1986; Brantl and Neubert, TIPS 7:6-7,1986; Glamsta et al., BBRC 184:1060-6, 1992; Teschemacher, Handbook Exp. Pharm. 104:499-28, 1993; Karelin et al., Peptides 16:693-7, 1995) as well as to peptides derived from combinatorial libraries screened for binding to opiate receptors (see Dooley et al., Peptide Research 8:124-137, 1995 for review). Each of these articles is hereby incorporated by reference.
The invention also includes a method of inhibiting or stimulating stem cell proliferation comprising contacting hematopoietic cells with a peptide selected from the group consisting of Tyr-MIF-1 related peptides, casomorphins, cytochrophins and exorphins. Specifically included are the Tyr-MIF-1 peptides having the sequences:
Tyr-Pro-Try-Gly-NH2 (SEQ ID NO:29),
Tyr-Pro-Lys-Gly-NH2 (SEQ ID NO:30),
Tyr-Pro-Leu-Gly-NH2 (SEQ ID NO:31), and
Pro-Leu-Gly-NH2.
The invention also includes a method of inhibiting or stimulating stem cell proliferation comprising contacting hematopoietic cells with an opiate peptide selected from the group consisting of
(D-Ala2,N-Me-Phe4,Gly-ol5)-Enkephalin (DAMGO),
(D-Arg2, Lys4)-Dermorphin-(1-4)-amide (DALDA),
(Phe4)-Dermorphine (1-4) amide
Ac-Arg-Phe-Met-Trp-Met-Arg-NH2, (SEQ ID NO:14);
Ac-Arg-Phe-Met-Trp-Met-Lys-NH2, (SEQ ID NO:32); and
H-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-NH2, (SEQ ID NO:33).
The invention also includes a method of inhibiting or stimulating stem cell proliferation comprising contacting hematopoietic cells with an opiate agonist compound selected from the group consisting of morphine, codeine, methadone, heroin, meperidine, alphaprodine, diphenoxylate, fentanyl, sufentanil, alfentanil, levorphanol, hydrocodone, dihydrocodeine, oxycodone, hydromorphone, propoxyphene, buprenorphine, etorphine, oxymorphone dextopropoxyphene, and meptazinol. Specifically included is morphine at inhibitory amounts less than 10xe2x88x927 molar.
The invention also includes a method of inhibiting or stimulating stem cell proliferation comprising contacting hematopoietic cells with an opiate antagonist or mixed agonist/antagonist selected from the group consisting of naloxone, naltrexone, nalorphine, pentazocine, nalbuphine and butorphanol. Specifically included is naloxone at inhibitory amounts of less than 10xe2x88x928 molar.
The invention also includes a method of stimulating stem cell proliferation comprising contacting hematopoietic cells with a stem cell stimulating amount of protein or peptide selected from the group that includes INPROL, myoglobin, DAMGO and DALDA.
The invention also includes a method of conducting gene therapy in a mammal comprising:
a) removing hematopoietic cells from said mammal,
b) treating said hematopoietic cells ex vivo with a stem cell stimulatory amount of INPROL and/or an opiate compound,
c) transfecting or infecting said hematopoietic cells with a predetermined gene,
d) contacting said transfected hematopoietic cells ex vivo with a stem cell inhibitory amount of INPROL and/or an opiate compound,
e) transplanting into said mammal the hematopoietic cells of step d
f) optionally treating said mammal in vivo with a stem cell inhibitory or stimulatory quantity INPROL and/or an opiate compound.
The invention also includes a method of conducting ex vivo stem cell expansion comprising treating said hematopoietic cells with stem cell inhibitory amounts of INPROL and at least one stimulatory cytokine. INPROL is contacted with the hematopoietic cells before, during and/or after contact with the stimulatory cytokine. Ex vivo stem cell expansion allows the production of sufficient amounts of stem cells from limiting sources such as cord blood, fetal liver, autologous bone marrow after chemotherapy, etc. or after purification (e.g., through fluorescent activated cell sorting using markers such as CD34, CD38 or rhodamine). The ability to selectively grow particular hematopoietic lineages also allows the clinician to specifically design stem cell transplants according to the needs of an individual patient.
The invention also includes a method of conducting ex vivo stem cell expansion comprising treating hematopoietic cells with stem cell stimulatory amounts of INPROL with or without at least one additional stimulatory cytokine. INPROL is contacted with the hematopoietic cells before, during and/or after contact with the stimulatory cytokine(s). Ex vivo, a stem cell stimulator will allow for expansion of stem cells and/or progenitors while a stem cell inhibitor will maintain stem cells in their undifferentiated state. The procedure can also be opdmized by the use of INPROL at stem cell inhibitory amounts in vivo to maintain stem cells in a quiescent state until they are engrafted, after which INPROL at stem cell stimulatory amounts can be used to stimulate bone marrow regeneration. Optionally, the hematopoietic cells may be split into two preparations and one treated with stem cell stimulatory amounts of INPROL to promote expansion of stem cells and/or progenitors while the other is treated with stem cell inhibitory amounts of INPROL to maintain stem cells in their undifferentiated state. The two preparations can then be combined and infused into a patient.
The invention also includes a pharmaceutical composition comprising (a) INPROL and (b) at least one inhibitory compound selected from the group consisting of MIP-1xcex1, TGFxcex2, TNFxcex1, INFxcex1, INFxcex2, INFxcex2, the pentapeptide pyroGlu-Glu-Asp-Cys-Lys, the tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro, and the tripeptide glutathione (Gly-Cys-xcex3Glu).
The invention also includes a pharmaceutical composition comprising (a) INPROL and (b) at least one stimulatory cytokine selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-13, EL-14, EL-15, G-CSF, GM-CSF, M-CSF, erythropoietin, thrombopoietin, stem cell factor, delta-like protein and flk2/flt3 ligand.
The current invention describes an inhibitor of stem cells (INPROL) which is different from those known in the art such as MIP-1xcex1, TGFxcex2, the tetrapeptide of Frindel and colleagues or the pentapeptide of Paukovits and coworkers (cf., Wright and Pragnell, 1992 (op. cit.)). Naturally occuring native INPROL has a molecular weight exceeding 10,000 daltons by ultrafiltration which distinguishes it from the tetrapeptide as well as the pentapeptide. It is more hydrophobic than MIP-1xcex1 or TGFxcex2 in reverse phase chromatography systems, distinguishing it from those cytokines. Further, its mode of action is different from that of any previously described inhibitor in that it is active in an in vitro assay when used during a preincubation period only. MIP-1xcex1 for example, is not effective when used during a preincubation period only (Example 5). Further, naturally occuring INPROL is active in an assay measuring xe2x80x9chigh proliferative potential cellsxe2x80x9d (HPP-PFC) whereas MIP-1xcex1 is not (Example 6). INPROL is different from those stimulators known in the art such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, G-CSF, GM-CSF, M-CSF, erythropoietin, thrombopoietin, stem cell factor, and flk2/flt3 ligand. Naturally occuring INPROL has little or no sequence similarity to these cytokines.